PROJECT SUMMARY Coronaviruses (CoVs) are a family of positive-sense RNA viruses that cause respiratory illnesses in humans ranging from the common cold to severe and lethal disease. The emergence of SARS-coronavirus (CoV) in 2002 and the continued circulation of MERS-CoV emphasize the capacity of CoVs to cause new zoonotic infections with pandemic potential. Despite the high mortality rates of these infections, no therapeutics or vaccines against any CoVs are currently available. Broadly active antiviral nucleoside analogs such as Ribavirin (RBV) are ineffective against CoVs. This limitation is attributed to a unique proofreading exoribonuclease (ExoN) in nonstructural protein 14 (nsp14-ExoN) that aids the RNA-dependent RNA polymerase (RdRp) encoded in nonstructural protein 12 (nsp12-RdRp) in high fidelity replication of these large positive-strand RNA viruses. We have identified two antiviral nucleoside analogs, GS-5734 and EIDD-1931, in collaboration with Gilead Sciences and the Emory Institute for Drug Development, respectively, which are broadly active against multiple CoVs with minimal cytotoxicity. In addition, we have identified two mutations within the predicted fingers domain of the nsp12-RdRp that reduce susceptibility to GS-5734, a C-adenosine nucleoside analog. The goals of this proposal are to define mechanisms through which these antiviral compounds inhibit CoV replication and determine the impact of resistance mutations on viral fitness, replication fidelity, and nucleotide selectivity. In Specific Aim 1, the basis of GS-5734 and EIDD-1931-mediated inhibition of CoV replication will be defined using deep sequencing, RT-qPCR, and Northern blot analysis to distinguish between the two most common mechanisms of antiviral action displayed by nucleoside analogs: chain termination and lethal mutagenesis. Experiments proposed in Specific Aim 2 will determine the impact of mutations that reduce susceptibility to GS-5734 and EIDD-1931 on coronavirus replication fidelity, viral fitness, and susceptibility to other nucleoside analogs. Together, these studies will probe mechanisms of GS-5734 and EIDD-1931 inhibition of CoV replication and explore the potential for these antiviral nucleoside analogs to individually and cooperatively serve as potent therapies against existing and emerging CoVs. This research also will inform the development of broadly active and complementary antiviral approaches to combat CoV infections. Finally, these studies will utilize GS-5734 and EIDD-1931 as tools to better understand mechanisms and viral mediators of CoV replication efficiency and fidelity.